The broad goal of this application is to characterize pathways of secretory trafficking in African trypanosomes using endogenous surface proteins (VSG and procyclin) as reporters. Trypanosomes cause disease in humans and livestock, and they avoid host immune responses by changing the expression of VSGs, the major glycosylphosphatidyl-inositol (GPI) anchored antigens of the bloodstream stage. Thus, proper trafficking of essential coat proteins, such as VSG, is critical to the success of the parasite. The interrelated Aims of this application all stem from observations made in the current funding period. First, trafficking of VSG and other reporters is critically dependent on GPI anchors, leading us to propose the GPI Valence Hypothesis: number of GPI anchors regulates progression and fate in the secretory pathway. Second, surface coat exchange (release of VSG and expression of procyclin) during differentiation involves both proteolysis and GPl hydrolysis of the old VSG coat. Proteolysis is mediated by a novel surface metalloprotease, and GPI hydrolysis unexpectedly involves surface expression of endogenous cytoplasmic GPI-PLC. Aim #1 will test the 'GPI Valence Hypothesis', that GPI valence controls progression, destination, and ultimately stability of membrane proteins within the secretory pathway of bloodstream stage parasites. Aim #2 will assess the role of membrane lipid rafts as the underlying mechanism of the Valence Hypothesis. Aim #3 will biochemically identify the metalloprotease (possibly a member of the newly identified MSP gene family), and will investigate the mechanism (thioacylation?) regulating surface expression of cytoplasmic GPI-PLC. Overall, these studies will illuminate essential secretory processes in trypanosomes and the role they play in the pathogenesis of trypanosomiasis.